Abstract
Purpose: :
The resistance to aqueous humor outflow across the trabecular meshwork (TM) is regulated by actomyosin contraction of its resident cells at the juxtacanalicular tissue. The phosphorylation of MLC, which elicits Myosin II ATPase activity, is regulated mainly by a Myosin II binding subunit (MYPT) of the myosin light chain phosphatase (MLCP) complex. In this study, we have examined the phosphorylation of MYPT by Rho kinase (ROCK), which induces actomyosin contraction.
Methods: :
Experiments were performed with a human glaucomatous cell-line (GTM3) cultured in 10% fetal bovine serum. Dephosphorylation of MYPT at Thr853 and Thr696 residues and the corresponding changes in ppMLC phosphorylation, induced by ROCK inhibitor Y-27632, were assessed using phospho-specific antibodies. Changes in cell adhesion to extracellular matrix were assessed in terms of impedance to current flow by electric cell substrate impedance sensing (ECIS) and IF to determine phosphorylation of paxillin (Tyr118) and FAK (Tyr397). The inhibitors effect on actomyosin contraction was quantified using collagen gel contraction (CGC) assay.
Results: :
The presence of serum led to significant phosphorylation of MYPT at both Thr853 and Thr696 (n=3). The extent of phosphorylation was reduced in a concentration-dependent manner at both the sites by Y-27632 (0.010 - 5 µM). The degree of inhibition was significant at Thr853 (IC50 of 0.6 µM compared to 2.2 µM for Thr696). Y-27632 also opposed MLC phosphorylation in a concentration-dependent manner, prevented serum-induced CGC, and reduced cell-substrate impedance at 4 kHz. Treatments with Y-27632 also reduced the stress fibers and tyrosine phosphorylation of paxillin, and FAK.
Conclusions: :
In TM cells, ROCK phosphorylation of Thr853 is the predominant site on MYPT, which leads to inhibition of MLCP and consequent actomyosin contraction. Since the Rho kinase inhibitors increase outflow facility, our findings indicate that their effect is possibly through modulation of cell-matrix interactions, which is strongly associated with the actin cytoskeleton.
Keywords: signal transduction: pharmacology/physiology • phosphorylation • trabecular meshwork